CHARACTERIZATION OF THE INTERACTION BETWEEN PCNA AND GADD45

We have examined the interaction between the DNA replication and repai r protein PCNA, and the growth arrest and DNA damage induced protein G add45. An anti-Gadd45 polyclonal antibody co-immunoprecipitates PCNA b ut in reciprocal experiments, an anti-C terminal anti-PCNA antibody fa iled to co-immunoprecipitate Gadd45, We used a yeast two hybrid assay to demonstrate that human Gadd45 interacts with both human and S. pomb e PCNA, We have determined that the N-terminal 94 amino acids of Gadd4 5 bind to PCNA, and using a series of N-terminal and C-terminal deleti ons of human PCNA we have mapped two potential Gadd45 binding sites, D eletion of the last 6 amino acids of PCNA ablated interaction, suggest ing a role in Gadd45 binding, This explains the inability of an anti-C terminal PCNA antibody to co-immunoprecipitate Gadd45, Using a peptid e ELISA approach, we showed that Gadd45 protein binds strongly to thre e regions of PCNA (residues 1-20, 61-80, and 196-215) and weakly to re sidues 121-170, The crystal structure of PCNA provides insight into ou r genetic and immunochemical data, Our results confirm an interaction between PCNA and Gadd45, define regions of both molecules involved in this interaction, and are consistent with a potential stoichiometry of 2 Gadd45 molecules to each PCNA monomer. These data provide support f or the notion that PCNA-Gadd45 interactions co-ordinate cell cycle and DNA repair.